Active membrane transport is a critical process for normal cell metabolism, including the maintenance of ion-gradients, osmotic balance, action potentials and apoptosis. The proposed work will address key questions regarding the mechanisms of nutrient uptake in Escherichia coli and other Gram negative bacteria. In E. coli, rare nutrients are sequestered by specific outer- membrane proteins that derive energy by coupling to the inner-membrane protein TonB. These TonB-dependent transporters include BtuB, which is responsible for vitamin B12 transport, and FhuA, FecA and FepA, which are responsible for the transport of various forms of chelated iron. TonB-dependent transporters are abundant in Gram negative bacterial and are critical to the success of many bacterial pathogens, such as the bacteria that result in meningitis, cholera and pertussis. Because they are unique to bacteria, these transporters are a logical target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for a number of TonB-dependent transporters; however, the mechanisms by which transport takes place is unclear. The proposed work will utilize site-directed spin labeling and EPR spectroscopy to test models for the molecular mechanisms of TonB-dependent transport, determine the mechanisms of transmembrane signaling and determine the mechanisms by which the transporter-TonB interaction is regulated. Finally, the structure and dynamics of these transporters (which are based upon 2-barrels) are influenced by both solute and lipid environment. Because of the critical need to generate and interpret high-resolution structural models of membrane proteins, the proposed work will also quantitate the influence of solutes and lipid environment on the structure of this class of membrane proteins. PUBLIC HEALTH RELEVANCE: The proposed research will determine the molecular mechanisms by which bacteria transport scarce nutrients across their cell membrane. This transport is critical to the survival of bacteria, and it is essential for the success of many bacterial pathogens, such as the bacteria that cause meningitis, cholera and pertussis. Knowledge of these transport mechanisms will assist with the development of new antibiotics that can inhibit bacterial growth.